Metastasis, or the spread of a primary tumor to secondary sites in the body, is the leading cause of death from cancer and effective anti-metastatic therapies are not currently available. The vacuolar (H+)-ATPase (V- ATPase) is an ATP-dependent proton pump that transports protons across intracellular and plasma membranes. It plays an essential role in intracellular, pH-dependent processes, including protein trafficking and degradation, endocytosis, and zymogen activation. V-ATPases present on the plasma membrane of specialized cells also function in such processes as bone resorption and pH homeostasis. It was recently discovered that the V-ATPase is expressed on the plasma membrane of invasive breast cancer cells and that inhibition of V-ATPase activity prevents in vitro invasion of these cells. Targeting of V-ATPases to different cellular membranes is controlled by subunit a, which in mammals is expressed in four isoforms (a1-a4). We have demonstrated that loss of subunit a3 in invasive breast cancer cells reduces invasiveness and its overexpression in a noninvasive breast cell line enhances both invasiveness and plasma membrane V-ATPase expression. We have recently found that the activity of plasma membrane V-ATPases is critical for breast cancer cell invasion. However, it has not yet been directly tested whether these plasma membrane V-ATPases contain subunit a3 and the mechanism by which the V-ATPase contributes to invasiveness is unknown. Furthermore, the role of the V-ATPase in in vivo breast cancer metastasis has not yet been assessed. Aim 1 of this project will determine whether subunit a3 is present in V-ATPases at the plasma membrane of a3- overexpressing breast cancer cells. Aim 2 will determine the mechanism by which the V-ATPase contributes to breast cancer cell invasion. I will test the hypothesis that invasive breast cancer cells target th pump to the plasma membrane to promote the extracellular acidification required for the maturation and activity of acid- dependent proteases involved in metastasis. Aim 3 will assess the role of subunit a3 in breast cancer metastasis in vivo. The results of these studies are criticl to exploring a3-containing plasma membrane V- ATPases as a novel and effective target to block breast cancer metastasis in vivo and will enhance our knowledge of the breast cancer metastatic cascade.